Culture Of Zygotic Embryos Research Articles

Statistical optimization of media for enhanced azadirachtin production from redifferentiated zygotic embryo cultures of neem (Azadirachta indica A. Juss.)

Callus cultures from zygotic embryos of neem (Azadirachta indica) were initiated and analyzed for azadirachtin production. Medium components were screened and optimized using the statistical techniques of Plackett-Burman and response surface methodology. The Plackett-Burman design, with five medium components (Murashige and Skoog major salts, sucrose, casein hydro- lysate, indole-3-acetic acid, and N 6 -benzylaminopurine), was performed to screen the variables that significantly affected azadirachtin production. The three variables— Murashige and Skoog major salts, sucrose, and N 6 - benzylaminopurine—significantly affected azadirachtin production and were significant factors for optimization using response surface methodology. The experimental results were fitted to a second-order polynomial model with a correlation coefficient (R 2 ) of 0.9582. The optimal concentrations of variables for maximum azadirachtin production were full-strength Murashige and Skoog major salts, 5.68% sucrose, and 10.42 μ MN 6 -benzylaminopurine. The maximum azadirachtin production by the predicted model was 5.13 mg/g dry weight, which was in agreement with the actual experimental value of 4.97 mg/g dry weight.

Expression analysis of somatic embryogenesis-related SERK, LEC1, VP1 and NiR ortologues in rye (Secale cereale L.)

The genetic basis of the regeneration process in cultured immature embryos of rye (Secale cereale L.) was analyzed. The experiments were designed to reveal differences between the in vitro culture responses of two inbred lines: L318 (a high regeneration ability) and L9 (a low potential for regeneration). The rye ortologues of plant genes previously recognized as crucial for somatic embryogenesis and morphogenesis in vitro were identified. Using oligonucleotide primers designed to conserved regions of the genes Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon 1 (LEC1), Viviparous 1 (VP1) and NiR (encoding ferredoxin-nitrite reductase), it was possible to amplify specific homologous sequences from rye RNA by RT-PCR. The transcript levels of these genes were then measured during the in vitro culture of zygotic embryos, and the sites of expression localized. The expression profiles of these genes indicate that their function is likely to be correlated with the in vitro response of rye. In line L9, increased expression of the rye SERK ortologue was observed at most stages during the culture of immature embryos. The suppression of ScSERK expression appears to start after the induction of somatic embryogenesis and lasts up to plant regeneration. The rye ortologues of the LEC1 and VP1 genes may function in a complimentary manner and have a negative effect on the production of the embryogenic callus. The expression of the rye NiR ortologue during in vitro culture reveals its importance in the process of plant regeneration.

Immature Zygotic Embryo Cultures of Arabidopsis. A Model System for Molecular Studies on Morphogenic Pathways Induced In Vitro

Immature Zygotic Embryo Cultures of Arabidopsis. A Model System for Molecular Studies on Morphogenic Pathways Induced In Vitro

PLOIDY VARIATION AND FLOWERING OF DWARF IRIS DERIVED FROM TISSUE CULTURE

Plant regeneration by tissue culture (somatic embryogenesis, organogenesis) has been achieved in zygotic embryo culture of dwarf iris (Iris pumila). Both regeneration processes are induced at the same time on MS nutritional media supplemented with 2,4-dihlorophenoxy acetic acid (2,4-D) at a concentration of 0.1-10 μM. White embryogenic callus is separated and cultured on medium with 2,4-D and kinetin (0.5 and 5.0 μM, respectively) resulting in somatic embryo formation. At the surface of green organogenic calli the shoots are formed. These shoots were for multiplication cultured on MS media supplemented with BAP and GA 3 (0.1 and 0.3 μM, respectively). This resulted in some flowering plants. In vitro formed flower parts have the same colour and morphology as the donor plants. Germination of somatic embryos as well as, rooting of shoots was achieved on MS medium without hormones. Plantlets regenerated by both processes acclimatized in greenhouse and flowered in next flowering season. Flow cytometric analyses revealed that most of regenerants have same genomic size while some alterations, especially among plants regenerated by somatic embryogenesis have also observed.

Arabidopsis tanmei/emb2757 embryo mutant is defective for in vitro plant morphogenesis

A mutation in the Arabidopsis TANMEI/EMB2757 (TAN) gene with an embryo defective phenotype was analysed for its effect on the morphogenic potential of somatic tissue cultured under in vitro conditions. The capacity for in vitro morphogenesis was evaluated using cultures of immature zygotic embryos, and seedling explants of the tan mutant and the parental Col-0 genotype. The explants were cultured on media supplemented with different plant growth regulators, and the capacity for two alternative pathways of morphogenesis, somatic embryogenesis (SE) and shoot organogenesis, was evaluated. Reporter genes (GUS, GFP) were used to monitor auxin and LEC2 and FUS3 gene activity in the tan explants. Moreover, the expression pattern of the TAN gene was analyzed during SE and in callus tissue of Col-0. It was indicated that the tan mutation resulted in a total lost of embryogenic and organogenic capacity of cultured tissues, suggesting the involvement of the TAN gene in basic cellular processes related to cell growth and differentiation. However, differential expression of the TAN gene during SE, and its increased activity at advanced stages of embryogenesis, implicate a specific role for the gene in the development of somatic embryos.

Sulfonylurea tolerance of wheat genotypes in zygotic embryo culture

Tolerance of wheat genotypes to the sulfonylurea herbicide metsulfuron-methyl was studied using in vitro culture. Six randomly selected wheat genotypes were used for isolation. Mature embryos were inoculated onto a modified MS medium to which three concentrations of metsulfuron-methyl were added: I-30 g l-1, II-60 g l-1, and III-90 g l-1. The control group of embryos was cultivated on a herbicide-free medium. After one month of cultivation, callus fresh weight was measured. There were significant differences among the genotypes regarding their tolerance to metsulfuron-methyl. The Pobeda cultivar expressed the highest and the Lr-12 genotype had the lowest level of herbicide tolerance.

Plant regeneration of southern Adriatic iris by somatic embryogenesis

A simple protocol has been developed for plant regeneration by somatic embryogenesis of Southern Adriatic iris (Iris pseudopallida Trinajstic), an endemic species of the Balkan Peninsula. Somatic embryogenesis was induced in zygotic embryo culture on media supplemented with 2,4-dichlorophenoxy acetic acid (2-10 mgL-1) as the sole plant growth regulator, where both embryogenic calli and somatic embryos were induced. Subsequent decrease of 2,4-D in the media promoted formation of somatic embryos. Developed somatic embryos germinated on medium without growth regulators. The regenerated plantlets had diploid chromosome number. Planted plantlets acclimatized very well under greenhouse and garden conditions.

Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.

Cultivo in vitro de embriões zigóticos e aclimatação de plântulas de coqueiro-anão

O objetivo deste trabalho foi ajustar os protocolos de cultivo in vitro de embriões zigóticos e de aclimatação de plântulas de coqueiro-anão-verde do Brasil de Jiqui (Cocos nucifera L.). Os embriões zigóticos maduros, colocados assepticamente em meio de cultura líquido Y3 suplementado com 27,8 mg L-1 de Fe2SO4.7H2O e 60 g L-1 de sacarose, apresentaram maior germinação e formação de plântulas normais. As plântulas transferidas para meio indutor de enraizamento Y3, gelificado e suplementado com 1 mg L-1 de ANA, 0,5 mg L-1 de BAP e 2,5 g L-1 de carvão ativado, apresentaram incremento do desenvolvimento radicular e da parte aérea. Na fase de aclimatação, o substrato composto por areia lavada e pó de casca de coco seco, na proporção de 1:1, proporcionou maior sobrevivência das plântulas (58,33%), maior crescimento da parte aérea (39,08 cm) e maior número de folhas (6,33). Os protocolos estabelecidos para o cultivo in vitro de embriões zigóticos e a aclimatação de coqueiro-anão-verde de Jiqui podem ser utilizados no intercâmbio e conservação de germoplasma.

Induction of somatic embryogenesis in Pinus heldreichii culture

The potential for somatic embryogenesis in zygotic embryo and megagametophyte cultures of Pinus heldreichii was examined. Somatic embryogenesis was initiated from megagametophytes containing immature zygotic embryos at early stages of development. An induction frequency of up to 6.7% was obtained on Gresshoff and Doy medium in the presence of 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l benzyladenine (BA). Formation and further proliferation of embryogenic tissue were achieved upon transfer of explants to a medium with reduced levels of growth regulators. Somatic embryos are being cultured for further development. .

Micropropagation of Tilia amurensis via Repetitive Secondary Somatic Embryogenesis

A optimal procedure for plant production via repetitive secondary somatic embryogenesis in Tilia amurensis is described. Somatic embryos were induced directly from the culture of zygotic embryos on medium with 1.0 mg/l 2,4.-D. Repetitive secondary somatic embryos formed on the surface of the cotyledons and hypocotyls except for the radicles when explants of somatic embryos were cultured on medium with 4.0 mg/l 2,4-D. The highest frequency of secondary embryo-genesis was obtained in the cotyledons (90%) and hypocotyls(83.33%) on MS medium with 1.0 mg/L 2,4-D. The average number of secondary embryos per explant was 25.74 in cotyledon and 24.92 in hypocotyl. When the cotyledon and hypocotyl segments from somatic embryos at different developmental stages were cultured on MS medium with 1.0 mg/L 2,4-D, the highest frequency of secondary embryogenesis was obtained from late cotyledonary secondary embryos. Somatic embryos were transferred to MS basal medium and then they germinated within 2 to 4 weeks of culture. Germinated somatic embryos grew normally into plantlets on WPM medium, producing new shoots. The converted plantlets were acclimatized on artificial soil mixture. These results indicate that the repetitive secondary somatic embryogenesis in T amurensis can offer the possibility to use in vitro culture system for the micropropagation.

IN VITRO REGENERATION OF ALLIUM AFLATUNENSE BY SOMATIC EMBRYOGENESIS

In vitro somatic embryogenesis and bulblet formation of Allium aflatunense in mature zygotic embryo culture were studied. Induction of morphogenesis was achieved on media supplemented with BDS mineral solution, 30 g L -1 sucrose, solidified by 0,7 mg L -1 agar and (in mg L -1 ): inositol 100.0, nicotinic acid 1.0, vitamin B 1 1.0, vitamin B 6 1.0, casein hydrolysate 100.0, L-proline 500.0, glicin 2.0, 2,4-D (2,4-dichlorophenoxyacetic acid, 2 mg L -1 ) or TDZ (thidiazuron, 2 mg L -1 ). Zygotic embryos cultured on BDS medium supplemented with 2,4-D formed friable embryogenic calli and some somatic embryos were formed. Somatic embryos were formed directly in the coleoptile and radicule region of zygotic embryos cultured on media with TDZ. The bulblet formation was also observed on BDS medium containing TDZ. Multiplication of somatic embryos, as well as, bulblets were observed after subsequent transfer on hormone free medium. Somatic embryos and bublets germinated after cold treatment (8 weeks) at +4°C.

EFFECT OF FRUIT CRYOPRESERVATION ON IN VITRO GERMINATION OF ZYGOTIC EMBRYOS OF SEVERAL SPECIES OF ILEX

This report presents preliminary results from a study aimed to develop a protocol for fruit cryopreservation and the recovering of plants through zygotic embryo culture, as a tool for the conservation of Ilex spp. (Aquifoliaceae) genetic resources. Fruits from eight plant species were harvested and maintained at 4°C for 1 month. Direct immersion of fruits in liquid nitrogen was lethal to the embryos of all the plant species tested. However, the embryos of five species (Ilex brasiliensis, L dumosa, I. microdonta, I. paraguariensis and I. pseudoboxus) dissected from fruits (with a cryoprotective solution containing 50% sucrose and 50% glycerol), cryopreserved using a slow freezing method (1°C.min -1 until -40°C) and stored in liquid nitrogen, when cultured in vitro, germinated with frequencies ranging from 3 to 23%. The embryos were cultured in vitro in darkness using a culture medium composed of 1/4-strength Murashige and Skoog medium, with 3% sucrose, 0.65% agar and 0.1 mg L -1 zeatin. The germinated embryos developed into normal plantlets after 60 days in culture. These results suggest that this methodology might be used for long-term germplasm storage of Ilex species.

IN VITRO PLANT REGENERATION IN ZYGOTIC EMBRYO CULTURE OF IRIS REICHENBACHII

Plant regeneration from mature zygotic embryo culture of Iris reichenbachii was studied. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D, 0-10 mg L -1 ) on induction of morphogenesis in vitro is investigated. The best response for callus induction is observed on MS medium with 10 mg L -1 2,4-D. White embryogenic, yellow non-embryogenic and green organogenic calli are observed. The fresh weight, as well as, percentage portion of embryogenic and organogenic calli is 2,4-D dependent. Shoots are succesfuly multiplied by adventitive organogenesis from organogenic callus. Germination of somatic embryos and rooting of shoots were achieved on MS solid medium without hormones.

IN VITRO MORPHOGENESIS IN ZYGOTIC EMBRYO CULTURES OF CAROB TREE (CERATONIA SILIQUA L.)

In this study, organogenic cultures were induced from zygotic embryos of carob tree. Basal medium consisted of Murashige & Tucker (1969), supplemented with 0.5 or 1 mg L -1 of 2,4-dichlorophenoxyacetic acid alone or combined with 0.5, 1, 2, 3 and 4 mg L -1 of kinetin. Calli initiation occurred in all the hormonal combinations tested. The zygotic embryo cultures differentiated two types of regenerants: somatic embryos (SEs) and shoots, with a maximum frequency of 30 and 100%, respectively. SEs exhibited morphological abnormalities, such as fusion and absence of cotyledons, and showed secondary embryogenesis. The regenerated shoots were rooted, hardened and transferred to the soil.

Hormone-response mutants of Arabidopsis thaliana (L.) Heynh. impaired in somatic embryogenesis

Plant hormones are considered to be the key factors involved in triggering in vitro induced plant morphogenesis, including somatic embryogenesis (SE). Mutants affected in SE and altered in hormonal response therefore provide valuable material for genetic research on in vitro induced plant embryogenesis. The capacity for SE was studied in 27 mutants with defects in response to different plant hormones: auxin, ABA, gibberellin and cytokinin, and evaluated in 2-week-old mutant and wild-type cultures in terms of their efficiency and productivity. SE was induced in vitro via a direct morphogenic pathway, through the culture of immature zygotic embryos on standard solid medium with 5 μM 2,4-D. The majority of the analyzed mutants displayed a significantly impaired capacity for SE; and those affected belonged to several different hormone-defective groups, including forms affected in auxin (axr4), gibberellin (ga) and ABA (abi, hyl1, cpb20, abh1) response. These mutants showed a significant decrease in embryogenic response as manifested by a low efficiency and/or productivity of SE. Additionally, SE efficiency was analyzed for axr4-1 mutant on media supplemented with different auxins while GA3 and inhibitors of gibberellins (uniconazol P and paclobutrazol), were applied for pkl1-1-mutant. The selected mutants provide a valuable research tool for studying the molecular mechanisms determining the induction of embryogenesis in cultures of somatic tissues. Their usefulness in further studies is discussed.

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl−1 sucrose. A sucrose concentration lower or higher than 30 gl−1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl−1 tyrosine in the medium. Seedlings germinated in the presence of 0.2–0.4 mgl−1 α-naphthaleneacetic acid and 0.3–0.5 mgl−1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60–70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.

In vitro screening for low temperature tolerance of wheat genotypes

Low temperature (LT) tolerance of 12 wheat (T. aestivum L.) genotypes was investigated in an in vitro zygotic embryo culture. Isolated embryos were grown on a modified MS medium for two months. Calluses were prepared by gradual decrease of temperatures (17?C-7 days, 5?C-7 days, -5?C-l day, -10?C-1 day) and then exposed to LT treatments (-15?C, 24 h and 48 h). After LT treatments, calluses were acclimated by gradual increase of temperatures and grown for another month. Based on the differences in callus fresh weight between the control group and calluses exposed to the LT treatments, levels of LT tolerance of wheat genotypes were determined. The results have shown that out of the 12 wheat cultivars, 6 had a high level of LT tolerance, 5 had a medium and only one spring cultivar (Venera) had a low level of LT tolerance. These results were compared with the results of the standard in situ test in a cold chamber. Only partial disagreement of results was determined in two cultivars with the medium level of LT tolerance (NSR-2 and Balkan). It can be concluded that the in vitro test can be successfully used for the separation of genotypes with high and low levels of LT tolerance, but for more precise separation within the medium level of LT tolerance, the test should be improved.

Phenol content related to antioxidant and antimicrobial activities of Passiflora spp. extracts

Methanolic extracts were prepared from different organs of plants from five Passiflora species obtained by zygotic embryo culture and evaluated for their capacity to quench DPPH and ABTS•+ radicals in comparison to that of Trolox, a water soluble vitamin-E analogue. Moreover their antimicrobial activity against E. coli was tested by agar diffusion and turbidity assays. P. nitida, P. foetida, and P. palmeri showed antimicrobial activity. P. nitida and P. palmeri also showed high antioxidant activity. P. tenuifila and P. coriacea demonstrated antioxidant power but not antimicrobial activity. The phenolic content of the different extracts was studied and quantified by spectrophotometric methods, HPLC, and mass spectrometry. High antioxidant activity correlated with high amounts of o-diphenol and catechin. An unknown component, tentatively identified as structural isomer of isoschaftoside, appeared to correlate with antimicrobial activity.

Improved efficiency of somatic embryogenesis from zygotic embryos in Hyoscyamus niger by seed water-soaking

We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somatic embryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the seeds in sterile distilled water for 16 h, prior to excision and culture of zygotic embryos, on MS basal medium supplemented with 2% sucrose, 2 g/l myo-inositol, 0.5 g/l 2-( N-morpholino) ethanesulfonic acid and 1 mg/l α-naphthalene acetic acid (NAA). The regenerated somatic seedlings were fertile and were morphologically uniform. This procedure is simple, rapid and effective for high frequency of plant regeneration via somatic embryogenesis. Moreover, this new method should facilitate the development of strategies to routinely transform recalcitrant plant species, including henbane.